Endolymphatic space is age-dependent

Knowledge of the physiological endolymphatic space (ELS) is necessary to estimate endolymphatic hydrops (ELH) in patients with vestibulocochlear syndromes. Therefore, the current study investigated age-dependent changes in the ELS of participants with normal vestibulocochlear testing. Sixty-four ears of 32 participants with normal vestibulocochlear testing aged between 21 and 75 years (45.8 ± 17.2 years, 20 females, 30 right-handed, two left-handed) were examined by intravenous delayed gadolinium-enhanced magnetic resonance imaging of the inner ear (iMRI). Clinical diagnostics included neuro-otological assessment, video-oculography during caloric stimulation, and head-impulse test. iMRI data analysis provided semi-quantitative visual grading and automatic algorithmic quantitative segmentation of ELS volume (3D, mm3) using a deep learning-based segmentation of the inner ear’s total fluid space (TFS) and volumetric local thresholding, as described earlier. As a result, following a 4-point ordinal scale, a mild ELH (grade 1) was found in 21/64 (32.8%) ears uni- or bilaterally in either cochlear, vestibulum, or both. Age and ELS were found to be positively correlated for the inner ear (r(64) = 0.33, p < 0.01), and vestibulum (r(64) = 0.25, p < 0.05). For the cochlea, the values correlated positively without reaching significance (r(64) = 0.21). In conclusion, age-dependent increases of the ELS should be considered when evaluating potential ELH in single subjects and statistical group comparisons.

It is necessary to be aware of asymptomatic ELH prevalence in healthy subjects to assess the relevance of any ELH found. Moreover, possible age-dependent physiological ELS changes should be considered on a single case or group scale. Unfortunately, however, studies on the ELH prevalence within the healthy population have been carried out noticeably less. Due to the more or less invasive nature of the methods able to verify ELH so far, studies on the healthy population to date were frequently replaced by patient groups that were thought not to be associated with ELH. At the same time, studies accompanied by comprehensive vestibulocochlear functional testing are scarce (for an overview cp. Tables 3 and 4). hTB studies often lack information on age and concurrent reference of vestibulocochlear function, while most iMRI studies are satisfied with the participants' statement that they did not perceive any vestibulocochlear malfunction. The comparison of the results of these studies is further complicated using different ELS quantification methods in iMRI [7] and different fixatives or post-mortem hours in hTB [23].
The current study investigated age-dependent changes in the ELS in participants with normal vestibulocochlear function ( vc HP) and discussed them given previous studies conducted on ELS in "healthy" participants.

Setting and institutional review board approval
All data were acquired at the interdisciplinary German Center for Vertigo and Balance Disorders (DSGZ) and the Munich University Hospital Neurology Department (LMU), Germany, between 2018 and 2020. Institutional Review Board approval was obtained before the initiation of the study (no. 641-15). Furthermore, all participants provided informed oral and written consent in accordance with the Declaration of Helsinki before inclusion in the study.

Study population
Thirty-two consecutive inpatients (64 ears; 20 females; aged 21-75 years, mean age 45.8 ± 17.2 years; 31 right-handed, two left-handed) of the Neurology Department without symptoms or underlying pathologies of the peripheral and central vestibulocochlear system underwent MRI with intravenous contrast agent as part of their diagnostic workup and agreed to undergo additional iMRI sequences after 4 h. Ethical considerations did not allow us to include healthy volunteers without a medical indication for an iMRI with contrast agent (see limitations for more information). The reasons for the participant's admission to the clinic included polyneuropathy (n = 5), movement disorders (n = 5), single small cortical metastases (n = 3), epilepsy (n = 3), optic nerve neuritis (n = 3), spinal inflammatory lesion (n = 3), tension headache (n = 3), viral meningitis (n = 2), subdural hematoma (n = 2), idiopathic facial nerve palsy (n = 2), and decompensated esophoria (n = 1). Cranial MRI findings were age-appropriate or did not interfere with the vestibulocochlear system. The laterality quotient for right-handedness was assessed with the 10-item inventory of the Edinburgh test [24]. The inclusion criterion was age between 18 and 85 years and normal audiovestibular testing to confirm the soundness of their peripheral end-organs and the central vestibular system (see Sect. 2.3). The exclusion criteria were current cochlear or vestibular disorders, a positive history of vertigo, balance, or hearing disorders, any MR-or contrast agent-related contraindications [25], poor image quality, or missing MR sequences.

Measurement of the auditory, semicircular canal and otolith functions
Participants without vestibulocochlear symptoms ( vc HP) underwent vestibulocochlear testing to confirm the soundness of their peripheral inner ear end-organs. Diagnostic workup included a thorough neurological examination (e.g., history-taking, clinical examination), neuro-orthoptic assessment [e.g., Frenzel glasses, fundus photography, and adjustments of the subjective visual vertical (SVV) for acute vestibular graviceptive dysfunction], video-oculography (VOG) during caloric and head impulse testing (HIT), and pure tone audiometry (PTA).
A tilt of the SVV is a sensitive sign of an acute graviceptive vestibular tone imbalance. SVV was assessed with the subject sitting upright in front of a half-spherical dome with the head fixed on a chin rest [26]. A mean deviation of > 2.5° from the true vertical was considered a pathological tilt of SVV [26].
The impairment of the vestibulo-ocular reflex (VOR) in higher frequencies was measured by HIT [24] using high-frame-rate VOG with EyeSeeCam ( [27], EyeS-eeTech, Munich, Germany). A median gain during head impulses < 0.8 (eye velocity in °/s divided by head velocity in °/s) was considered a pathological VOR [28].
Furthermore, semicircular canal responsiveness in lower frequencies was assessed by caloric stimulation (CS) with VOG, which was performed for both ears with 30° cold and 44° warm water. Vestibular paresis was defined as > 25% asymmetry between the right-and left-sided responses [29] or the sum of the maximal peak velocities of the slow phase caloric-induced nystagmus for stimulation with warm and cold water on each side < 25°/sec [30]. The caloric asymmetry index (AI CS ) was calculated based on the slow-phase PTA was based on both ears' four-tone average (arithmetic mean) of the thresholds at 0.5, 1, 2, and 3 kHz. Hearing loss was defined as PTA > 25 dB [31]. In all tests, the contralateral ear was masked by adequate noise. All audiometric equipment is regularly recalibrated (every 6 months) according to the local university equipment standard.

Data acquisition
Participants received a standard dose (0.1 mmol/kg body weight) of gadobutrol (Gadovist ® , Bayer, Leverkusen, Germany) once and were scanned in a whole-body 3 Tesla MRI scanner (Magnetom Skyra, Siemens Healthcare, Erlangen, Germany) with a 20-channel head coil twice; first directly after intravenous injection, as part of their diagnostic workup, and a second time after 4 h for the iMRI sequences. A T2-weighted, 3D-FLAIR (three-dimensional fluid-attenuated inversion recovery) sequence was used to differentiate endolymph from perilymph and bone, and a spin-echo 3D-SPACE (three-dimensional sampling perfection with application-optimized contrasts using different flip angle evolutions) sequence delineated the total inner ear fluid space from the surrounding bone according to the method suggested by Ref. [2].

Semi-quantitative (visual) grading of the endolymphatic space
ELH was observed as enlarged negative-signal spaces inside the labyrinth and cochlea on the 3D-FLAIR images [2]. Semi-quantitative (SQ) visual grading of the ELS (sqELS) was performed independently by an experienced head and neck radiologist (SSt) and a neurologist (JG, VK) who were blinded to the clinical patient data. The ELS's characterization in the vestibulum and cochlea was based on a previously described 4-point [7,32] ordinal scale classification.

3D-(volumetric) quantification of the endolymphatic space.
3D-or volumetric quantification of the ELS (vELS) was achieved in two steps: First, segmentation of the total fluid space (TFS) was based on IE-Vnet [33], a recently proposed and pre-trained volumetric deep learning algorithm with V-net architecture. IE-Vnet was deployed via the TOMAAT module [34] into the 3D-Slicer toolbox (version 4.11, [35]).
Second, ELS and perilymphatic space (PLS) were differentiated within the TFS using volumetric local thresholding (VOLT, [36]) that uses ImageJ Fiji [37], including the "Fuzzy and artificial neural networks image processing toolbox" [38] and the "MorphoLibJ Toolbox" [39]. The resulting 3D volume included ELS and PLS classifications for cochlea and vestibulum (cutoff 6). The ELS ratio, ER[%] = ELS TFS × 100 , was calculated analogously to Ref. [40]. ELS symmetry between both inner ears was assessed using the absolute value of the asymmetry index AI

Statistics and validation parameters
Statistical analyses were performed using the Statistical Package for Social Sciences software (SPSS, Inc, Chicago, IL, USA). Categorical values are reported as the number of cases that fit the category/number of participants with normal vestibulocochlear testing [%]; ordinal or scalar values are presented as (mean ± standard deviation). Results were reported at a significance level of p < 0.05 and p < 0.01. In addition, the linear agreement between parameter pairs was calculated using two-sided Pearson's correlation coefficient.

Results
A detailed clinical and neurophysiological characterization of the group of participants with normal vestibulocochlear testing ( vc HP) is provided in Table Table 2.
In the following, the mentioned results will be discussed given previous studies conducted on ELS in "healthy" participants.

ELH prevalence in "healthy" participants
The results of the current study fit well with the findings in the literature. An overview of ELH prevalence in studies on the "healthy" population to date is shown in Table 3. Notably, even excluding studies using the non-affected ear in unilateral Meniere's disease (uMD) as a healthy control group, a relatively high ELH prevalence was found. In iMRI studies (see Table 3b) using semi-quantitative threepoint visual grading [41], mild ELH was reported to lie between 3.3 and 28.6% of participants in the cochlea [10,42], 6.7-25% in the vestibulum [7,43], and 7.5-10% in the inner ear [7,43]. Significant ELH was reported between 9.5 and 13.3% in the cochlea [10] and up to 30% in the vestibulum [42]. In contrast, in hTB studies' adult population (see Table 3a), including participants with MD, ELH prevalence was found to be around 9% [44], and without MD, around 4.5% [6].
In summary, the overlap of the 3-point sqELH grading system of Schuhknecht et al. [45] for the hTB studies and of Nakashima et al. [41] for the iMRI helps to compare their results, a direct transfer of the results should be done with caution. After all, while iMRI can only indirectly verify ELH, it provides in-vivo longitudinal ELS visualization with multi-slice 3D-quantification in addition to contemporary measurement of vestibulocochlear function. hTB studies, on the other hand, provide direct identification of the distension of Reissner's membrane and further histopathologic evidence for MD.

ELS volume in "healthy" participants
The current study's ELS volume was low in the cochlea 2.8 ± 1.1 mm 3 , vestibulum 6.6 ± 2.1 mm 3 , and inner ear 9.4 ± 2.3 mm 3 compared to volumes in the previous literature, especially across methods in both hTB [23,46,47] and highresolution CT [48][49][50]. A detailed description of the ELS 2D-(area) and 3D-(volumetric) quantification in previous hTB and iMRI studies on the "healthy" population to date can be viewed in Table 4.
It is noticeable that the lower volumetric values or ratios occur in the studies with participants that were controlled for vestibulocochlear testing. In addition, the absolute values of both ELS quantification methods (although 2D-more than the 3D-ELS quantification method) are variable across the different studies, and relative size ELS are less variable (with ELS-Ratio being most constant, also across methods). Table 3 Literature overview of ELH prevalence in "healthy" participants This table has no claim to be exhaustive. Inclusion criteria for this table were the investigation of "healthy" controls and the usage of an established, comparable method for the semi-quantitative classification of the endolymphatic hydrops (sqELH). For the hTB studies, the 3-point sqELH grading system of Schuhknecht et al. [45] was used. For the iMRI studies, the 3-point sqELH grading system of Nakashima et al. [41] was used. Here, "healthy" control includes patients without audiological or vestibular problems and excludes studies using the non-affected ear in unilateral Meniere's disease as a healthy control group + = including patients with Morbus Meniere, MD; ■ = significant ELH following sp 3 [41]; ☐ = mild ELH following sp 3 and sp 4 ; ∆ = using the human temporal bone collection at the Massachusetts Eye and Ear Infirmary; A = adult population including normal ears and cases with otological diseases (for more detail see [6]); c HP = participants with nonotological diseases (for more detail, see [10]) without audiological, vestibular or neurological problems; vc HP = participants with neurological diseases (for more detail, see [7]) with normal vestibulocochlear testing; c/ oVEMP = ocular/cervical vestibular evoked myogenic potential; CS = caloric stimulation; HP = healthy participants with no history of audiological, vestibular, or neurological problems (for more details, see [43]); HIT = head-impulse test; I = infant population including congenital anomalies (51.4%), infectious diseases (17.1%), non-infectious diseases (18.6%), and tumors (12.9%); N = newborn mainly premature population; n. s. = not specified; PTA = pure tone audiometry; sq 3 = semi-quantitative following 3-point classification in cochlea and vestibulum following [41]; sq 4 = semi-quantitative following 4-point classification in cochlea and vestibulum following [7], sq h = semi-quantitative following 3-point histological classification of the cochlea following [45]; SVV = subjective visual vertical; TFS = total fluid space; V = visual assessment

Table 4
Literature overview of previous ELS 2D-and 3D-quantification results in "healthy" participants This table has no claim to be exhaustive. Inclusion criteria were the investigation of "healthy" participants and the usage of an established, comparable method for a two-dimensional (2D) or three-dimensional (3D) quantification of the endolymphatic space (ELS). tological diseases (for more detail, see [46]) and without audiological, vestibular or neurological problems; vc HP = participants with neurological diseases (for more detail, see [7]) with normal vestibulocochlear testing; CRS = chronic rhinosinusitis; CS = caloric stimulation; EcochG = electrocochleography, ELS = endolymphatic space; ER = ELS ratio or 3D ELS/TFS in [%]; HP = healthy participants; it = intratympanic iMRI; iv = intravenous iMRI; HIT = head-impulse test; hTB = human temporal bone study; M = manual measurement using software; MRI = magnetic resonance imaging; n. s. = not specified; o/cVEMP = ocular/cervical vestibular evoked myogenic potential; SVV = subjective visual vertical; TFS = total fluid space; V =

ELH laterality and ELS symmetry
In healthy participants, cochlear and vestibular ELH laterality (bi-or unilateral) and ELS symmetry (when using 2D or 3D quantification) were omitted. Investigated hTB and iMRI studies either only have one ear at their disposal [46,[51][52][53], limit information on laterality to the patient cohort [6, 10, 42-44, 54, 55], or do not discuss ELH laterality as a category [40,[56][57][58]. The current study showed 12.5% unilateral mild ELH in the cochlea and vestibulum, 9.4% showed a mild bilateral ELH in the cochlea, and 18.8% a mild bilateral ELH in the vestibulum, while the asymmetry index remained < 25% (cochlea: 20.2%, vestibulum 15.2%, inner ear: 12.7%). Laterality and symmetry add to the characteristic pattern in "healthy" participants, alongside volumetric extent and maximally mild ELH in semi-quantitative ELS quantification. Therefore, ELS studies should include semi-quantitative (SQ) grading and 3D quantification. SQ grading should include the 3-point grading scale by Nakashima et al. [41] as it is the most commonly used grading system and can serve as a calibration point. Reported 3D-quantification values should include symmetry parameters, such as asymmetry indices for un-normalized data and the relative size of the ELS for normalized data.

ELS age-dependency
Age and vELS were found to be significantly positively correlated for the inner ear (r(64) = 0.33, p = 0.008), and vestibulum (r(64) = 0.25, p = 0.045). For the cochlea, values correlated positively without reaching significance (r(64) = 0.21, p = 0.100) (Fig. 1). These results seem plausible because of the increased vestibulocochlear dysfunction with age [59][60][61][62], including the frequent cases without the patient noticing the vestibular dysfunction occurred [63][64][65]. Contrary, two published studies by Inui et al. [40,58] showed that participants < 60 years had significantly larger TFS and ELS/TFS volume ratios in the inner ear and significantly larger ELS/TFS volume ratios in the vestibulum in comparison to the participant group ≥ 60 years. The differing results between the current study and the latter might be explained by the difference in methods or the selection of participants (Table 4b).
The overall mild degree in ELS increase without symptoms and (in general) normal audiovestibular testing results also further question the current definition of ELH. The word hydrops implies an excessive pathologic accumulation of endolymphatic fluid within the ELS. However, the current results imply that a mild ELH might not necessarily be a pathologic sign but can represent a physiological norm, especially with increasing age and when unaccompanied by audiovestibular symptoms or (diagnostic) signs of audiovestibular dysfunction. An objective description of different ELS distention grades without statement on its pathogenicity (± ELH) would be preferable. An easy solution would be to simply retain all previous suggestions of semi-quantitative ELH grading as ELS grading, and discard the statement referring to no, mild, moderate or severe ELH.

Methodological limitations of the study
Two major limitations of the current study need to be considered: first, the limited number of subjects included, and second, the circumstance that iMRI imaging could only be done in patients with other (neurological) pathologies. A representative ELS study with a higher number of completely healthy subjects, ideally of 20 participants per decade, ranging between 20 and 90 years of age, would be desirable and is still pending. Second, influences of the participants' underlying neurological pathologies on the ELS appear unlikely (view inclusion and exclusion criteria, Sect. 2.2.) but cannot entirely be excluded. In addition, two of our participants showed signs of presbyacusis in our audiometric testing, and we do not know if hearing performance influences the ELS. However, ethical considerations did not allow us to include healthy volunteers without a medical indication for an iMRI with contrast agent. The decision to avoid unnecessary contrast agent application was based on prior findings of signal intensity increases in the dentate nucleus and globus pallidus on T1-weighted MR images after applying MR contrast agents that are still under investigation [66][67][68]. Third, the study lacks histological confirmation of endolymphatic hydrops since the in-vivo acquisition of histological specimens is currently not possible.

Conclusion
Age-dependent increases of the ELS should be considered when evaluating potential ELH in single subjects and statistical group comparisons. LMU (Association for the Promotion of Science and Research at the LMU Medical Faculty), and the German Federal Ministry of Education and Research (BMBF) via the German Center for Vertigo and Balance Disorders (DSGZ, grant number 01 EO 0901).

Conflicts of interest
There are no conflicts of interest.
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